Abstract: Alphaviruses are vector-borne pathogens that have proven capable of causing significant outbreaks of clinical disease. Currently, there is an absolute lack of safe and approved antiviral therapies / vaccines. Thus, research which defines the molecular basis of infection is essential and critically important to not only understanding alphaviral pathogenesis, but to the development of innovative antiviral strategies. The overarching research objective of this proposal is to define the mechanism by which a novel alphavirus RNA species impacts infection and pathogenesis. The scientific premise and rationale supporting this objective are based upon a wealth of preliminary evidence which indicates that the production of noncapped genomic RNAs (ncgRNAs) is essential for alphaviral infection, as evidenced by studies involving Sindbis virus (SINV) and Ross River virus. Nonetheless, the extent to which the ncgRNAs impact viral infection is unknown, and represents a critical gap in the knowledgebase. Thus, we propose the following specific aims- i) To determine the mechanism with which the noncapped genomic vRNAs impact infection, and ii) To define the impact of the noncapped genomic vRNAs on viral pathogenesis. To accomplish the first aim we will undertake an exhaustive molecular analysis of SINV infection to determine the mechanism by which the ncgRNAs effect viral infection. Specifically, we will determine whether or not ncgRNA function is linked to its translatability via a novel IRES-based approach. In addition, we will determine which viral process is negatively affected by ncgRNA reduction; and determine the molecular interactions required for ncgRNA function. The second aim uses a combinatorial approach of in vivo studies and IFN-competent tissue culture models to comprehensively define the contribution of the ncgRNAs to SINV pathogenesis. Briefly, the contribution of the ncgRNAs to the induction of a detrimental neuroinflammatory response will be evaluated in depth histologically, and at the transcriptomic and proteomic levels. Moreover, the consequences of increasing and decreasing alphaviral capping efficiency on the host innate immune response will also be defined in highly tractable tissue culture models of infection. The proposed work is highly significant in that it seeks to characterize and define the molecular importance of a novel viral RNA species. Moreover, the proposed research is conceptually and technically innovative as state-of-the-art techniques, approaches, and models of infection are used to delineate the contributions of the novel ncgRNA species to molecular infection and pathogenesis. The successful completion of this work will fundamentally expand the knowledgebase by eliminating the critical gap in understanding described above; in addition, the proposed research has a high likelihood of leading to the identification of novel antiviral strategies.